Edited by James C. Liao, Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, and approved November 5, 2019 (received for review June 28, 2019)
Efficient microbial synthesis in challenging pathways relies on dynamic regulation of multiple metabolic fluxes to balance several competing goals. To address these situations, we developed an autonomous, pathway-independent, and layered regulation tool. By incorporating parts from 2 different quorum-sensing systems, the layers of our system can be tuned independently to ensure generalizability. Application of the regulation system to overcoming 2 different sets of challenges in the naringenin and salicylic acid pathways resulted in significant improvements in titer, demonstrating that the system is an effective tool for improving pathway production.
Metabolic engineering seeks to reprogram microbial cells to efficiently and sustainably produce value-added compounds. Since chemical production can be at odds with the cell’s natural objectives, strategies have been developed to balance conflicting goals. For example, dynamic regulation modulates gene expression to favor biomass and metabolite accumulation at low cell densities before diverting key metabolic fluxes toward product formation. To trigger changes in gene expression in a pathway-independent manner without the need for exogenous inducers, researchers have coupled gene expression to quorum-sensing (QS) circuits, which regulate transcription based on cell density. While effective, studies thus far have been limited to one control point. More challenging pathways may require layered dynamic regulation strategies, motivating the development of a generalizable tool for regulating multiple sets of genes. We have developed a QS-based regulation tool that combines components of the lux and esa QS systems to simultaneously and dynamically up- and down-regulate expression of 2 sets of genes. Characterization of the circuit revealed that varying the expression level of 2 QS components leads to predictable changes in switching dynamics and that using components from 2 QS systems allows for independent tuning capability. We applied the regulation tool to successfully address challenges in both the naringenin and salicylic acid synthesis pathways. Through these case studies, we confirmed the benefit of having multiple control points, predictable tuning capabilities, and independently tunable regulation modules.
Author contributions: C.V.D. and K.L.J.P. designed research; C.V.D. performed research; C.V.D. analyzed data; and C.V.D. and K.L.J.P. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1911144116/-/DCSupplemental.
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